Proposal Summary/Abstract The innate immune system utilizes restriction factors to prevent integration of foreign RNA species, such as viral RNA, into the genome of the host cell. In response, viruses adapt mechanisms to evade restriction in their natural hosts. An example of this type of molecular arms race is that between APOBEC3 restriction factors and HIV-1 infectivity factor, Vif. The proposed mechanism of restriction of HIV-1 by the APOBEC3 family involves several steps. First, APOBEC3 proteins associate with host and viral RNAs and are incorporated into budding virions. After the core contents of the new virus particle are released into a nave host cell, the APOBEC3 enzymatic activity targets viral ssDNA for deamination of cytidine nucleotides. HIV-1 counteracts APOBEC3 proteins with Vif, which recognizes APOBEC3 proteins of its natural host and recruits the host ubiquitin ligase machinery to target these proteins for proteasomal degradation. Typical APOBEC3 restriction factors (A3D, A3F, and A3G) are comprised of two homologous zinc- binding domains with distinct roles in cellular restriction activity. One domain carries the enzymatic deaminase function, while the other domain is poised to interact more readily with RNA for packaging of APOBEC3 molecules into new virions. Vif-interacting regions of APOBEC3 restriction factors can be localized to either domain. APOBEC3H (A3H) is particularly fascinating because, while other APOBEC3 restriction factors require two domains to facilitate all functions, A3H uses one, highly multifunctional domain, thus, making it an ideal, minimal system to study restriction by APOBEC3 restriction factors. Interestingly, experiments to understand adaptations during infection in a pig-tailed macaque model revealed that the A3H was one of the most highly polymorphic restriction factors. Several polymorphic variants of A3H displaying a wide range of restriction activity were identified in the infected animals. These natural variants provide an innovative opportunity to characterize various functions of A3H on the molecular level and relate these biochemical findings to cellular phenotypes. This research proposal aims to characterize A3H as a model to dissect restriction activities of APOBEC3 proteins. This research will focus on human A3H (haplotype II) in combination with polymorphic variants of pig- tailed macaque A3H. The following properties of A3H will be characterized experimentally through biochemical assays and crystallographic analyses of 1) A3H 3D structure, 2) cytidine deamination activity, 3) RNA binding and effect on catalysis, and 4) Vif binding and affinity. Biochemical activities will be correlated to restriction activity in cells. These experiments will lead to a more detailed understanding of HIV-1 restriction by A3H.